The long range goal of this research program is to understand mechanisms of regulation in multisubunit proteins and other macromolecular assemblies. We are interested in elucidating the physical bases whereby individual molecular components operate in concert as systems to produce and control biological functions, approach is to study processes of protein-protein interactions and protein-ligands interactions in specific systems by (a) characterizing the components and the interactions in thermodynamic and kinetic terms and (b) relating these properties to specific molecular structures of the interacting molecules, and to their known biological functions. Components of the program include: (a) development of new methods for the study of interacting systems and (b) theoretical work on the thermodynamics of linked processes in macromolecular assemblies. Through work funded by this grant we recently achieved a significant breakthrough by developing methods to study the functional energetics of hemoglobin tetramers in all eight intermediate states of heme-site ligation. this has opened the door to obtaining previously-inaccessible knowledge regarding properties of the intermediate state species and their roles in the cooperative mechanism. Initial studies indicate that each tetrameric molecules acts as a three-level molecular switch to control ligand affinity; a specific distribution of the ten ligation state species among the three cooperative levels defines a "code" for the molecular switching mechanism. By extending these studies to encompass a wider range of conditions (pH, temperature, DPG, C1, and other heme-site ligands) we will determine how general the effects are, and how the distribution of free energy states (and their enthalpic and entropic components) are controlled by the physiological regulatory species. We plan to combine these studies with the use of mutational altered amino acid residues as thermodynamic reporter groups to map the pathways of cooperative free energy transduction within the hemoglobin tetramers. This research program is now at its most exciting stage: we are on the verge of being able to actually deduce the rules of molecular switching in the hemoglobin control system. This will constitute an unprecedented advance of singular importance to the field of regulatory protein assemblies.